KR101081809B1 - Method of deleting HIV-1 5'LTR/gag gene using ginseng - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for HIV-1 5'LTR and / or gag gene defects using ginseng as an active ingredient. The pharmaceutical composition of the present invention can induce a deletion of the HIV-1 5'LTR and / or gag gene by administering to an HIV infected individual. Therefore, the present invention can be usefully used for the prevention and treatment of AIDS.

Ginseng, red ginseng, HIV-1, 5'LTR, gag gene, deficiency

Description

Method of deleting HIV-1's 5'LTR / gAg gene by processing ginseng {Method of deleting HIV-1 5'LTR / gag gene using ginseng}

The present invention relates to a method of processing the ginseng to delete the 5'LTR / gag gene of HIV-1.

HIV-1 (Human Immunodeficiency Virus-1) is the pathogen of the Acquired Immunodeficiency Syndrome ("AIDS"). HIV-1, along with HIV-2, belongs to the lentiviridae of the retrovirus, and the infectious virion has an RNA (+) genome consisting of two identical single-stranded. When HIV-1 infects a cell, the RNA is converted into double-stranded DNA by reverse transcriptase, which is encoded by the RNA genome, which is inserted into the host cell's chromosome. This results in the formation of a provirus. The proliferation of the virus in the proviral state transcribs the viral genes, producing the mRNAs of the proteins necessary for replication and assembly.

The HIV-1 genome (9.2 kb) is a structural gene of gag, pol, env and accessory genes such as tat, rev, nef, vif, vpu, vpr, and long terminal repeats at both ends. sequence) (see FIG. 8).

The gag and pol proteins are made by decomposing precursor proteins, the env protein becomes gp160 precursor protein, and is broken down into gp120 and gp41 during assembly. The first infection of HIV is asymptomatic or manifests as a cold-like condition. The virus enters a latent state due to latent infection in CD4 T cells, macrophages, microglial cells, etc. Antibodies to gp120 appear 3-20 weeks after infection, after which time the infection can be diagnosed. During the incubation period of 2-10 years, changes in the immune system are induced, and prodromal period symptoms (AIDS-related complex (ARC) -fatigue, fever, weight loss, diarrhea) appear before the immunodeficiency symptoms. When the number of CD4 T cells falls below 200 / μl due to HIV infection, symptoms of immunodeficiency appear and are clinically defined as AIDS.

Panax ginseng C. A. Meyer (Panax schinseng Nees) is a perennial root of the genus Ginseng of the Araliaceae family, and has been used as an adjunct to the treatment of various diseases in Asian countries for thousands of years. Ginseng has been tested through many pharmacological experiments to reduce cholesterol, inhibit lipid peroxidation, lower blood pressure, increase blood flow, expand cerebrovascular system, increase cardiac function, antiarrhythmic, antithrombotic, chronic renal failure, immune control, memory increase, brain metabolism. Anti-inflammatory, anti-stress, antioxidant, anti-aging, anti-ulcer and gastric secretion, anti-diabetic, detoxification, hepatocellular enzymes, asthma treatment, anti-inflammatory, analgesic, anemia treatment, increased fertility, decreased alcohol blood levels, anti It is known to have activities such as allergy and anticancer drugs.

In addition, ginseng and red ginseng are classified according to the processing method, ginseng is a so-called raw ginseng not processed, dried ginseng, dried ginseng is called ginseng. Red ginseng (Red Ginseng) is steamed ginseng dried and processed to have a water content of 14% or less. Brown ginseng is promoted during the manufacturing process.

Therefore, ginseng containing about 75% moisture is difficult to preserve for a long time and ginseng components can be decomposed by various enzymes contained in the ginseng itself due to microbial contamination during distribution. Reduction of moisture through the prevention of bacteria, fungi and microorganisms, and has the advantage of easy storage and transport by reducing the volume and weight. Saponin quantitative method and quality control, which is the representative active ingredient of ginseng, prevented the degradation of saponin during the processing process, and saponin, maltol, ginsenoside Rh2 and other active ingredients were additionally produced. That is, the main component of ginseng is saponin, and red ginseng is processed so that its efficacy is not altered.

In particular, red ginseng has a sedative and arousing effect on the central nervous system. It also acts on the circulatory system to prevent hypertension or arteriosclerosis, while reducing hematopoietic and blood sugar levels and protecting the liver. It acts indirectly on sexual behavior or reproductive effects by acting on the endocrine system, has anti-inflammatory and anti-tumor effects, protects against radiation and protects the skin. And softens. In addition, the most important of the effects of red ginseng is the adaptogen (adaptogen) effect, which increases the ability of the body to adapt to various harmful effects from the environment, such as fistula (각종), various stresses, etc. to make the body more easily adaptable. . To date, numerous studies have been conducted at home and abroad on the components and pharmacological effects of red ginseng, and especially red ginseng has been reported to have antioxidant activity (Korean Ginseng Journal, Kwon Yong-hoon, 24 (1), 29-34, 2000). .

In addition, the inventors also found that in previous studies, Korean red ginseng (KRG) alone in patients with HIV-1 infection, ^1-4 , or ⁵ , along with zidovudine (ZDV) after 1991, were very potent antiretroscopy. bar has a bar ^6, treatment with these drug therapy (HAART, highly active antiretroviral drug therapy ). ^{1 through 7,} running speed Any significant inverse between (progression rate) related to the treatment KRG and AIDS (AIDS) moves through the clinical application was ^8. The mutation rate of the HIV-1 gene is inversely related to KRG treatment. Moreover, KRG treatment has slowed the development of resistance to zidovudine. According to the results of the inventors, KRG treatment slowed down the reduction of CD4 T cells regardless of the therapy or other characteristics of the patients. Recent analyzes of combination therapy with KRG and HARRT have shown that consistent viral control is possible with these combinations without the occurrence of drug resistance mutations ⁷ .

The cause of the slow progression of AIDS in long-term nonprogressors (LTNPs) or long-term surviviors (LTSs) in the cohorts of the present inventors will be described here. Recently, we have reported a relationship between the rate of large-scale deficiency of nef gene and KRG intake time in 10 LTS patients receiving continuous KRG treatment ⁹ . Compared to the products amplified in the nef gene from these 10 long-term survivors, the genes amplified in the gag and 5'LTR regions showed a high proportion of two bands (wild-type and shortened bands). appear.

Although there have been many studies on ^10-18 nef gene large-scale deletions in LTNP, only a few cases have reported the association of large-scale defects in the gag and 5'LTR regions to date. More specifically, one LTS infected with HIV subtype C reported a deletion of one of three amino acid codons in the gag gene ¹⁹ . In another study, two of 24 inexperienced individuals showed a 16bp deletion and a small 9bp deletion in 5'LTR ²⁰ . However, no possible association has been reported between long-term progression or slow progression of HIV infection and genetic defects in the 5'LTR and gag regions.

Accordingly, the present inventors completed the present invention by identifying deficiencies of 5'LTR and gag genes of HIV-1 when red ginseng was administered to HIV-1 infected patients for a long time while studying a method of treating AIDS.

REFERENCES

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28. Keum YS, Han SS, Chun KS, Park KK, Park JH, Lee SK, et al .: Inhibitory effects of the ginsenoside Rg3 on phorbol ester-induced cyclooxygenase-2 expression, NF-kappaB activation and tumor promotion. Mutation Res . 2003; 523-524: 75-85.

29.Kim KH, Lee YS, Jung IS, Park SY, Chung HY, Lee IR, and Yun YS. Acidic polysaccharide from Panax ginseng, ginsan, induces Th1 cell and macrophage cytokines and generates LAK cells in synergy with rIL-2. Planta Med. 1998; 64: 110-115.

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It is an object of the present invention to provide a method of treating ginseng to delete the 5'LTR / gag gene of HIV-1.

In order to achieve the above object of the present invention, the present invention provides a pharmaceutical composition for HIV-1 5'LTR / gag gene deletion comprising ginseng as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention is characterized by the fact that ginseng ingestion may delete HIV-1 5'LTR / gag gene which is closely related to virus growth and AIDS etiology.

Preferably, the ginseng is characterized in that the red ginseng.

In one embodiment of the present invention, to determine whether there is a relationship between the occurrence of large-scale defects (gΔ) and KRG intake in the 5'LTR and gag regions, we treated 10 years of treatment with KRG (13,364 ± 5,364g) for more than 12 years. The long-term surviviors (LTSs) and 8 long-term survivors (controls) who did not consume or consumed a minimum of 1,436 ± 1,027 g of Korean red ginseng were examined for the 1,125 sequences surrounding these parts. In these 10 LTSs, 189 PCR products were obtained from 80 peripheral blood mononuclear cells (PBMC) samples, with 44 (55%) of the total 80 PBMCs and 71 (37.6%) of 189 PCR products being massively defective. (gΔ) was shown. 55% of PBMCs and 37.6% of PCR products were seen in 10 LTSs, while the values of 8 control LTSs were 30.3% and 14.8%, respectively. These differences are statistically significant (P <0.05 and P = 0). In addition, the ratio of defective genes in 28 general control patients who did not take red ginseng was 13,3% and 8.3% based on PBMC and PCR products, respectively (see Example 3, FIGS. 1 to 3).

Thus, administration of red ginseng to individuals can lead to deletion of the HIV-1 5'LTR / gag gene, which can be useful for the prevention and treatment of AIDS.

Red ginseng used as an active ingredient of the pharmaceutical composition of the present invention can be prepared by a known method.

In general, red ginseng is prepared through a steaming step of steaming steamed ginseng (about 75% of moisture) with steam under constant temperature conditions and a drying step of drying the steamed ginseng. The red ginseng extract may be prepared by heating and extracting the red ginseng thus prepared or concentrated to red ginseng concentrate.

Meanwhile, in the present invention, the red ginseng extract or concentrate may be used alone or in addition to one or more pharmaceutically acceptable carriers, excipients or diluents, and then formulated in a conventional manner, and then used. Acceptable refers to compositions that are physiologically acceptable and do not normally cause an allergic reaction, such as gastrointestinal disorders, dizziness, or the like when administered to a human. In addition, the red ginseng of the present invention formulated as above may be administered through a suitable route of administration. Suitable routes of administration may be oral administration.

Preferably it may be formulated using methods known in the art in the form of preparations for oral administration such as powders, granules, tablets, pills, dragees, capsules, solutions, gels, slurries, suspensions and the like. For example, oral formulations can obtain tablets or dragees by combining the active ingredients with solid excipients and then grinding them, adding suitable auxiliaries and processing them into granule mixtures. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol and starch, cellulose, including starch, corn starch, wheat starch, rice starch and potato starch, etc. Fillers such as cellulose, gelatin, polyvinylpyrrolidone, and the like, including methyl cellulose, sodium carboxymethylcellulose, hydroxypropylmethyl-cellulose, and the like. In addition, crosslinked polyvinylpyrrolidone, agar may be optionally included. , Alginic acid or sodium alginate and the like can be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like.

In addition, red ginseng as an active ingredient of the pharmaceutical composition of the present invention can be purchased commercially. In one embodiment of the present invention was used red ginseng powder capsule sold by Korea Ginseng Corporation.

The pharmaceutical compositions of the present invention can be administered to mammals, especially animals, including humans. Preferably, it may be administered in an effective amount to the patient infected with HIV-1. Said "effective amount" refers to an amount effective to delete the nef gene of HIV-1 in vitro or in vivo.

The effective amount of the pharmaceutical composition according to the present invention is a specific use described by those skilled in the art in consideration of various tolerances such as route of administration, frequency of treatment, age, weight, health condition, sex, severity of disease, diet and excretion rate of the subject in need of treatment. The appropriate effective dosage can be determined according to. Preferably, the effective amount of the pharmaceutical composition comprising red ginseng of the present invention is 50-100 mg / kg / day for men and 20-70 mg / kg / day for women.

The pharmaceutical composition of the present invention can be administered continuously to delay progression to AIDS disease after diagnosis in a subject.

On the other hand, the defect of the 5'LTR and / or gag gene in the present invention may be defined as having a premature stop codon, lack of an initiation codon.

In one embodiment of the present invention, genetic defects such as non-methionine start codons, early maturation stop codons by G-to-A hypermutation, and small defects were observed from amplification products generated from 10 LTSs. (Example 4). In each patient and 10 LTS, the size of the deletion, duplication or insertion was all different. In eight control LTSs, genetic defects due to premature stop codon and non-methionine start codons were observed (FIG. 4).

Preferably, the 5'LTR and / or gag gene deleted by the method of the present invention may be represented by the following sequence: GenBank accession No. EF 370172, EF 370173, EF 370175, EU 047612, EF 370184, EF 370186, EF 370187, EF 370 188, EF 370 191, EF 370 192, EF 370193, EF 370 196, EF 370197, EF 370 199, EF 370 201, EF 370 200, EF 370 200 , EF 370 205, EF 370 207, EF 370 211, EF 370 213, EF 3702 115, EF 3702 116, EF 3702 118, EF 370 219, EF 370 221, EF 370 223, EF 370 224, EF 370 225, EF 370 226, EF 370 227, EF 370 228, EF 370 231 370235, EF 370237, EF 370239, EU 047647, EF 370245, EF 370 250, EF 370 251, EF 370 252, EF 370 253, EF 370 256, EF 370259, EF 370 262, EF 370261, EF 370 265, DQ 295196, EF 370275, EF 370 276 EF 370277, EF 3702778, EF 370279, EF 370282, EU 047673, EF 370283, EF 370285, EF 370286, EF 370287, EF 370288, EF 370292

As described above, the pharmaceutical composition of the present invention can induce deletions in HIV-1 5'LTR and / or gag genes by administering to HIV infected individuals. Therefore, the present invention can be usefully used for the prevention and treatment of AIDS.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are provided to illustrate the present invention, and the present invention is not limited to the following examples and may be variously modified and changed.

&Lt; Example 1 >

Subject and KRG Treatment

The study group consisted of 10 long-term survivors (LTSs) who received maximum KRG, 8 control LTSs, and 28 HIV-infected patients who had never taken KRG.

Ten LTSs in this study were described in detail in our previous study ⁹ . All 10 were diagnosed between 1987 and March 1996 and no antiretroviral treatment was administered in this study, except for one patient (93-04). The patient received intermittent ZDV (200 mg or 300 mg daily dose) from April 1993 to August 1997.

Eight control LTSs were selected from our cohorts. Patients classified as control LTS were diagnosed with HIV-1 infection prior to 1996, and they were given a minimum dose (1,526 ± 1,183 g) of KRG or none (patient) in the absence of very potent antiretroviral medication. 89-05) KRG was not administered. The only patients 91-23 received ZDV from April 1993 to August 1999. Patients 89-05 were removed from the study sample of the present invention in 1997 because the patient went to another country. Of the eight control LTSs, two recipients in the blood (long-term survivors 91-20) (patients 91-22 and 91-23) were included. Patients 89-26 and 92-23 were reluctant to consume KRG. Patient 90-01 was a plasma donor who sold 83 plasma. The patient was described as provider O ²¹ . Therefore, the baseline characteristics were similar when comparing 10 high KRG intake LTS with 8 control LTS.

In other words, there was no significant difference in basal disease progression or CD4 T-cell number between 10 LTS and 8 control LTS, only difference in KRG intake.

In addition, another 28 control patients were investigated. Of these 28, 87-05 and 90-50 wives were included. Some of these 28 patients were long-term survivors. But they were not included in the long-term survivor group, since the diagnosis was made after 1997. Obtained informed consent from each participant.

KRG treatment in HIV-1 infected patients began in NIH in Korea in late 1991. The KRG used for the experiment used red ginseng powder capsules sold by Korea Ginseng Corporation. The daily dose of KRG for each patient was 5.4g for men and six 300mg capsules three times a day and 1.8g for women. The duration of KRG administration for each patient is shown in FIG. 1.

The average amount of Korean red ginseng administered to 10 LTS was 13,364 ± 5,364g for 155 ± 28 months, and 1,436 ± 1,027g for 132 ± 29 months in 8 control LTS (FIG. 5). The annual amounts of KRG in 10 LTS and 8 control LTS were 1,035g and 131g, respectively. There was a statistically significant difference of 7.9 times in KRG intake level (P <0.001).

<Example 2>

CD4 T Cell Count and Plasma HIV-1 RNA Copy Level

Blood was drawn from each patient of Example 1 at intervals of 3 to 6 months, and peripheral blood mononuclear cells (PBMC) were phycoerythrin (PE)-and fluorescent isothiocyanate (FITC) in each sample. Each was incubated with an antibody against the conjugated CD4 antigen (Simultest reagent; Becton Dickinson, San Jose, CA, USA). Levels of CD4 T cells were measured using a FACScan (Becton-Dickinson) flow cytometry. HIV-1 RNA concentration in plasma was measured with Amplicor HIV-1 Monitor kit (Roche Diagnostics, Branchburg, NJ) ⁷ .

The number of CD4 T cells was measured at month 6 for 6 months in 10 LTS and 8 control LTS fed Korean red ginseng, and the results are shown in FIG. 1.

 The number of CD4 T cells was significantly decreased from 245 ± 160 / μl initially measured to 555 ± 208 / μl after 168 ± 29 months in 10 LTS patients receiving maximum Korean Red Ginseng treatment (22 / μl reduction per year, FIG. 1A). To FIG. 1J). In addition, the 8 control LTSs were first reduced from 574 ± 222 / μL to 353 ± 297 / μL after 132 ± 29 months (20 / μL reduction per year, FIGS. 1K-1R).

In both groups, CD4 T cells were found to be reduced in a similar fashion over 10 years.

<Example 3>

Partial 5'LTR Site and Gag Amplification

Proviral DNA was extracted from the uncultured PBMCs obtained in Example 2. first round primer CE23 (5'-tgtggatctaccacacacaaggctactt-3 '; 46 to 73; SEQ ID NO: 1) and 514 (5'-tccagaatgctggtagggtatac-3'; 1,617 to 1,640; SEQ ID NO: 2), second round primer CE1 (5'-cgagagctgcatccggagtacta -3 '; 297 to 319; SEQ ID NO: 3) and 512 (5'-ctgcagcttcctcattgatggtc-3'; 1,398 to 1,420; SEQ ID NO: 4) and third round primer 501 (5'-gtgtggcctgggcgggactg-3 '; 380 to 399; sequence No. 5) and No. 297 to 1,421 by double- (rarely triple-) nested PCR using 510 (5'-gatgtaccatttgcccctgga-3 '; 1,202 to 1,222; SEQ ID NO: 6), the numbering, here and below, is PCR products of 1,125 bp up to derived from the sequence of HIV NL4-3) were amplified from each sample. (The positions 297 to 1,421 are the partial 5'LTR and the gag gene.)

The maximum response performed at one time for each sample, including the negative control, was four. On the other hand, for the amplification of the nef gene, the maximum response was 5 at one time for each sample, including the negative control. PCR products were purified and sequenced immediately. PCR contamination was monitored by physical separation of PCR products following BLAST search, amino acid codon comparison by manual alignment, and phylogenetic analysis.

The experimental results are expressed as mean ± standard deviation. Statistical significance was assessed by Student's two-tailed t-test and Chi-square test using SPSS package version 12.0.

As a result, 189 PCR products were amplified from 80 PMBC samples recovered from 10 LTSs at different time points (FIGS. 1A to 1J). At many time points all 10 LTS showed gΔ at the 5′LTR / gag gene site (FIG. 1). Defects were observed in 44 of the 80 samples (55%) (FIG. 2). Of the 189 PCR products, 71 (37.6%) showed gΔ 5′LTR / gag. Of these, 23 (32.4%) amplification showed only one short band without the wild-type gene. The characteristics of the 71 gΔ5′LTR / gag are summarized in FIG. 7.

Five PCR products (obtained from patients 87-05 in January 2007, 89-17 in July 2001, 90-05 in October 1993, and 92-13 in February 2006 and May 2004.) And two PCR products (Δ86bp from 90-05 patients in October 1993 and Δ420bp from 90-50 patients in 2006) contained gΔ in the 5'LTR site or in the gag gene, respectively (Table 3). In the remaining PCR products, the deletions were located at both terminal portions of the 5'LTR (including at least 753 position; Δ37bp) and at the beginning of the gag gene (Δ29bp at position 818 for HIV-1 NL43). In other words, the deletion was more than 66 bp near the gag gene initiation codon.

Thus, the ratio of gΔ in the 5'LTR fraction was 36.5% (69/189) and the gΔ ratio based on the gag gene was 34.9% (66/189). It can be said that KRG administration affected 5'LTR more than gag gene.

Apart from patients 91-20 and 93-60, the initial observation of gΔ at the 5'LTR and gag sites was faster than the deficiency in the nef gene after initiation of KRG intake from patients where no sample was available ⁹ . The median time point from the onset of KRG for the first gΔ observation was 26 months (19 months to 101 months); This is shorter than the time required to observe defects in the nef gene ^9.

Patients 87-05 were co-infected with HIV-1 subtype B and HCV derived from contaminated blood coagulation factor 9 produced overseas. This patient began administering KRG in June 1994. Although there was a small deletion of 9 bp in all amplifications, among 10 LTSs, it did not show the only gΔnef ⁹ . Surprisingly, we observed gΔ in the earliest useful samples obtained in April 1997, and a total of 7 gΔ from 36 PCR products obtained from 4 of 12 samples (FIGS. 1A-1J, FIG. 5). This patient has not had any acquired immunodeficiency-related symptoms since April 2007, which is the first case to survive for more than 20 years in Korea without any antiretroviral therapy.

 Patients 89-17 started KRG administration in December 1991. And compliance with the administration was inconsistent. One gΔ reported in July 1993 was truly exceptional. This is because Δ917bp was replaced by the terminal portion of the 474 bp and gag genes (2,029 bp to 1,838 bp) of the pol gene (backwards 4,296 to 4,037 of NL4-3). The patient started HARRT in February 2002 and had a CD4 T cell count of 513 / μL in April 2007.

Patients 90-05 were diagnosed with HIV-1 infection in February 1990 and began to receive KRG in December 1991. The patient showed the best compliance with KRG treatment in this study. The patient consumed his own personal KRG with a supply of 21,522 g of KRG. The daily dose of KRG was 6.0 g (10 300 mg capsules twice daily) with 95% compliance. The first gΔ observation was January 2001 (111 months after the start of KRG administration, KRG intake was 12,012 g in total), and gΔnef was 87 months earlier than the first observed (FIGS. 6A, 6B, 6C). An example of gΔ and detailed information on the remaining seven LTSs are shown in FIGS. 1 and 6.

In addition, 88 PCR products were amplified from 33 PBMC samples recovered from 8 control LTS. 13 gΔ (14,8% of amplified) were present in 10 PBMC samples (30.3% of the total). Ten LTSs contained a significantly higher percentage of gΔ in both PBMC samples and PCT products than the eight control LTSs. (P <0.05 for the former, P <0.0001 for the latter) (FIG. 3).

In addition, 60 PCR products were amplified from 30 PBMC samples obtained regardless of clinical stage from 28 patients who did not receive KRG. 5 gΔ (8.3% of amplified) were present in 4 PBMCs (13.3% of total).

<Example 4>

Genetic defects and minor defects

In addition to large-scale defects, we observed genetic defects such as non-methionine start codons, early maturation stop codons by G-to-A hypermutation, and small defects. Of the 10 LTSs, patients 87-05 had premature mature stop codons in four sequences (EU047601, EU047605, EU047607, and EU047609), and patients 89-17 also had premature mature stop codons and start codons (GenBank EF370193). Isoleucine. Patients 90-18 had a 6bp insertion in the gag gene, while patients 92-13 had two duplications of 102 bp (GenBank EF370252), a 159bp deletion in the 5'LTR region, and a G to A overmutation. (Genbank EF370256) and also had a 6bp insertion between 327 and 328 parts (GenBank EF370258) (Figure 4).

Of the eight control LTSs, patients 91-22 had isoleucine as the start codon in early mature stop codons or in three sequences (GenBank EF370314, EU047654, and EU047655). Patients 89-26 had two premature mature stop codons in one sequence (EU047635).

Example 5

Frequency of large-scale defects according to the number of CD4 T cells

The present inventors analyzed the frequency of large-scale defects according to the number of CD4 T cells. Above all, 10 LTS PCR amplification products were grouped into CD4 T cell number 200 / ul or less, 200-400 / ul, 400 / ul or more. The incidence of large-scale defects in 10 LTSs administered with KRG was 47.4% (9/19), 51.8% (14/27), and 61.8% (21/34), which was slightly different for the number of CD4 T cells. Can present dependencies However, in 8 control LTS, the proportion of large defects was 44.4% (4/9), 20% (4/20), 50% (2/4), and 18.2 in 28 HIV-1 positive patients without KRG. % (2/11), 14.3% (1/7), 8.3% (1/12).

<Discussion>

The inventors have continued to pay attention to the health of LTS or LTNP in Korea, and as a result, in spite of the absence of beneficial host factors such as 32bp deletion in CCR5 or HLA-B57 in Koreans, Many LTSs were observed among the 323 HIV-1 infected people diagnosed with.

More specifically, nine HIV-1 infected patients were diagnosed in Korea in 1987, three of which were still alive, and all three had KRG combined with one treatment or HARRT. Long-term survivors 87-05 are one of these patients who have been taking KRG since June 1996 (FIG. 1A). Another two of these patients received HARRT from 1998 to 1999 with KRG, and their CD4 T cell counts were 231 / ul in 1998, 6 / ul 1999 from 1,052 / ul December 2004, 2006 It increased to 1,127 / ul each year.

In addition, 22 HIV-1 patients were diagnosed in 1988 and only two patients (9%) have been treated with KRG. This patient was not included in the study of the present invention because the patient's HIV-1 subtype is CRF02_AG, but the patient was classified as LTNP with a CD4 T cell count greater than 500 / ul. Many patients diagnosed after 1997 combined HARRT with KRG, and they showed the absence of drug resistance mutations, which showed a surprisingly good response to this combination therapy.

In a previous study, we have reported that KRG treatment is associated with a higher frequency of ⁹ major nef gene defects in HIV-1 infected patients and slows disease progression ^1,8 .

In the present invention, which in spite of ever Empty ⁹ than four PCR products amplified from the nef gene bar, hayeotneun amplify the three PCR products from each sample, and in 10 LTS same inventors, nef deletion (34.3% of the sample and 18.8% of the PCR product) a large deletion (was observed a higher rate of 37.6%) in 55% of the samples, the PCR product compared to the ⁹ (P <0.0001). The frequency of deletion of 5'LTR / gag was 1.6 times / 2.0 times higher than that of nef gene deletion. 61 of the total 80 samples had previously been applied to nef gene amplification, and 13 of the 61 samples (21.3%) showed massive deletions in both genes. In particular, patients 87-05, 89-17, 90-05, 90-50, and 96-51 showed significantly higher frequency of major defects in the 5'LTR / gag gene than in the nef gene (P <0.05). (FIG. 5). In addition, the proportion of samples showing a large deficit was found to be 37.6% in 10 LTS treated with KRG, which was significantly higher than in 8 control LTS (14.8%) and 28 HIV-1 infected patients (8.3%). Ever higher. In addition, it was confirmed in the present invention that there was no relationship between the frequency of massive defects and the number of CD4 T cells in any of the 8 control LTS or other 28 patients (Example 5). The relationship between deficits and KRG intake is supported.

The findings show a modest dependency on the CD4 T cell number and a good comparison in 10 LTS. Among the 10 LTS treated with KRG, patients 87-05, 90-05, 92-13, and 96-51 who showed consistent good loyalty maintained CD4 T cell counts (FIGS. 1A, 1C, 1G, 1J), whereas Patients who did not show good loyalty in the last 1-2 years had a rapid decrease in the number of CD4 T cells (FIGS. 1E, 1F, 1I). As described above, the decrease in the number of CD4 T cells by attitudes has already been shown in patients 89-17 and 90-18 (FIGS. 1B and 1D). Since 10 years, a long time without any AIDS-related symptoms, many patients tended to be less concerned about their health or to ignore KRG intake.

Recent papers have revealed the genetic characteristics of full-length viral sequences in LTNP. Alexander et al. ²² found no defects in genes other than the nef gene, and found early maturing stop codons, out-of-frame deletions and total defects. Unusual polymorphism and large nef gene defects were seen in 8 LTNPs. Huang et al. ^23,24 also reported genetic defects in 5'LTR and gag genes in 8 LTSs. The entire viral sequence was obtained in this study, and G to A hypermutations in the 5'LTR and gag genes were found in only one patient. Recently, Calugi et al. Identified a large deficit in the env gene (the gene next to the nef gene) ²⁵ . Blankson et al. ²⁶ showed viral loads of plasma RNA of less than 50 copies / mL, and no large-scale deletions were observed in the HIV-1 full-length sequence of the supressor.

HIV-1 gene expression is regulated by several very highly conserved cis-acting regulatory elements in the TATA box and LTR containing the binding sites of the transcription factors NF-kB and Spl ²⁷ . Some components are known to inhibit NF-kB and Ap-1 transcription factor activity in human cell lines ²⁸ . The LTR moiety is more likely to be affected by KRG uptake than in other parts of HIV-1.

In the present invention, a large deficit of 5'LTR was observed in 10 LTSs, including patients 87-05 (which did not show the nef gene massive deficiency). Moreover, the size of each observed large defect was large, but was located only at a single site within the gene. The present inventors have identified HIV-1 full-length sequencing of large-scale deletion of the env gene (> 1,000 bp) in six LTSs (patients 90-05, 90-18, 90-50, 91-20, 92-13 and 96-51). It was confirmed when it was obtained. (Data not shown)

Thus, irrespective of the relationship between KRG uptake and genetic defects, although the observation of the present invention is only one finding among the various beneficial factors observed in the present study, the results of the present invention result in a large part of the deficiency in the gag and provide the primary evidence that HIV-related disease and significant progress ^1-9. Regarding possible mechanisms of how viral gene deletions affect LTS or LNTP, we estimate that the gene deletions are the result of indirect immune modulation by ingestion of KRG. Ginseng is well known to mediate cell-mediated immune response (cell mediated immune response) by the Th1 cytokines and immune suppression generally active (generalized immune activation state) in ^29-32, HIV-1 infection. Nevertheless, it is thought that KRG intake is not the only factor that LTS without KRG still has a higher 5'LTR / gag deficit than in other 28 patients infected with HIV. This finding supports the importance of genetic defects induced by KRG in prolonging the life of LTS or LNTP.

<Sequence DATA>

178 of the 336 nucleotide sequences were submitted to GenBank, and GenBank accession No. EF370172 in EF370172, EU047667 in EU047600, EU047676 in EU047670 and EU047693 in EU047681.

As noted above, the pharmaceutical compositions of the present invention can induce deletions in HIV-1 5'LTR and / or gag genes by administering to HIV infected individuals. Therefore, the present invention can be usefully used for the prevention and treatment of AIDS.

1 shows changes in CD4 T cell counts in 10 LTSs (FIG. 1 a-j) with minimal KRG or 8 control LTSs (FIG. 1 k -r) and maximum KRG without antiretroviral treatment. Korean red ginseng administration period is indicated using bar. Three PCR reactions were performed to amplify one sample, the samples used for the 5'LTR and gag gene sequences, and the samples indicating the large 5'LTR and gag gene defects, respectively, indicated by the down and up arrows at the bottom. It became. In both groups, CD4 T cell numbers decreased over 10 years. So there was no significant difference between the two groups except for the KRG dose.

Figure 2 shows the analysis of 5'LTR and gag sequencing in peripheral blood monocytes obtained from the earliest three LTS diagnosed. The amplified product was isolated by electrophoresis on 1% agarose gel. PCR products from patients 87-05, 89-17 and 90-05 are shown in lanes 1-10, 11-18 and 19-31, respectively. Patients 87-05 had four wild types (lanes 1,3,6,9) with five overlapping bands (lanes 2, 7, 10) and five single short bands (lanes 4, 8). Had a short band Patients 89-17 displayed seven short bands (lanes 11, 12, 13, 14, 16-18), of which lane 14 was one short band (14%). Patients 90-05 showed seven short bands (lanes 19, 22, 23, 24, 26, 29, 30). Lanes 22, 24, 26, 29, and 30 showed one short band (71%).

(M: marker, WT: wild type band)

Figure 3 compares the frequency of major defects among the three groups. Percentage of peripheral blood monocyte samples indicating a large deficit was higher in KRG LTS (55%) than in 2 28% of nine control LTS as well as another 28 patients (13.3%). In addition, the proportion of PCR products positive for massive defects was significantly higher in 10 LTS-administered KRGs than in another 28 patients (8.3%) as well as 13.9% of the eight control LTSs (37.6%). . Of the 28 patients, 87-05 and 90-50 wives were included. In addition, some of them were less than 10 years after HIV-1 diagnosis, but were likely to become LTS.

4 shows the expected amino acid sequence of a representative partial gag protein from 10 LTS infected with HIV-1. In the United States, the consensus amino acid sequence of gag protein from non-progressive patients is shown in the top row (Ref. 23), and the expected sequence for 10 LTS is as follows. Dots indicate sequence identity, and bars indicate partial or large defects in addition to the variant. And a question mark means premature mature stop codons. Amino acid sequences are represented by one letter code. The source of each sequence was identified by each patient code (the first two digits represent the year of HIV diagnosis and the two digits following the hyphen indicate the unique number assigned to each patient in Korea). The date of the sample for each GenBank number is shown in FIG. In FIG. 7, seven base sequences without a deletion in the gag gene were not seen.

5 is a table showing the clinical characteristics of 18 LTS infected with HIV-1.

Except for the past year, compliance with patients 91-20 of LTS was best with 90-05, and his actual KRG dose was over 22,422 g by personal purchase. Patients 91-22 and 91-23 were HIV-1 infected from blood provided from 91-20. Except for LTS 87-05, all patients were infected with Korean subclade of HIV-1 subtype B.

Figure 6 shows the characterization of large-scale deletion of the 5'LTR / gag gene in 18 LTS and 28 KRG non-administered patients.

Figure 7 shows the frequency of large-scale deletions in the 5'LTR / gag gene compared to the large-scale deletions in the nef gene.

<110> KOREA GINSENG CORP.          University of Ulsan Foundation For Industry Cooperation <120> Pharmaceutical composition for deleting HIV-1 5'LTR / gag gene          configuring ginseng <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> first round primer CE23 <400> 1 tgtggatcta ccacacacaa ggctactt 28 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> first round primer 514 <400> 2 tccagaatgc tggtagggta tac 23 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> second round primer CE1 <400> 3 cgagagctgc atccggagta cta 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> second round primer 512 <400> 4 ctgcagcttc ctcattgatg gtc 23 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> third round primer 501 <400> 5 gtgtggcctg ggcgggactg 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> third round primer 510 <400> 6 gatgtaccat ttgcccctgg a 21  

Claims (6)

Treatment of ginseng in animals other than humans to delete 5'LTR (long terminal repeat), gag or 5'LTR and gag genes of HIV-1 (human immunodeficiency virus-1) with the sequence shown below: GenBank accession No. EF 370172, EF 370173, EF 370175, EU 047612, EF 370184, EF 370186, EF 370187, EF 370 188, EF 370 191, EF 370 192, EF 370193, EF 370 196, EF 370197, EF 370 199, EF 370 201, EF 370 200, EF 370 200 , EF 370 205, EF 370 207, EF 370 211, EF 370 213, EF 3702 115, EF 3702 116, EF 3702 118, EF 370 219, EF 370 221, EF 370 223, EF 370 224, EF 370 225, EF 370 226, EF 370 227, EF 370 228, EF 370 231 370235, EF 370237, EF 370239, EU 047647, EF 370245, EF 370 250, EF 370 251, EF 370 252, EF 370 253, EF 370 256, EF 370259, EF 370 262, EF 370261, EF 370 265, DQ 295196, EF 370275, EF 370 276 EF 370277, EF 3702778, EF 370279, EF 370282, EU 047673, EF 370283, EF 370285, EF 370286, EF 370287, EF 370288, EF 370292 The method of claim 1, wherein the ginseng is red ginseng.      In vitro, HIV-1 is treated with ginseng to delete 5'LTR, gag or 5'LTR and gag genes of HIV-1 with the sequence shown below: GenBank accession No. EF 370172, EF 370173, EF 370175, EU 047612, EF 370184, EF 370186, EF 370187, EF 370 188, EF 370 191, EF 370 192, EF 370193, EF 370 196, EF 370197, EF 370 199, EF 370 201, EF 370 200, EF 370 200 , EF 370 205, EF 370 207, EF 370 211, EF 370 213, EF 3702 115, EF 3702 116, EF 3702 118, EF 370 219, EF 370 221, EF 370 223, EF 370 224, EF 370 225, EF 370 226, EF 370 227, EF 370 228, EF 370 231 370235, EF 370237, EF 370239, EU 047647, EF 370245, EF 370 250, EF 370 251, EF 370 252, EF 370 253, EF 370 256, EF 370259, EF 370 262, EF 370261, EF 370 265, DQ 295196, EF 370275, EF 370 276 EF 370277, EF 3702778, EF 370279, EF 370282, EU 047673, EF 370283, EF 370285, EF 370286, EF 370287, EF 370288, EF 370292 delete delete delete

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